Interferon-mediated inhibition of production of Gazdar murine sarcoma virus, a retrovirus lacking env proteins and containing an uncleaved gag precursor.

HTG2 cells are murine sarcoma virus-transformed hamster cells. These cells continuously produce Gazdar sarcoma virus particles which are devoid of viral envelope proteins and which contain the uncleaved gag precursor polyprotein, Pr65, as their major protein constituent. Human interferon-alpha elicited an antiviral response in these cells as shown by the inhibition of replication of vesicular stomatitis virus in interferon-treated cells. Extracellular production of the retroviral particles by these cells was also inhibited by interferon in a dose-dependent manner and this inhibition was abolished by a specific antiserum to interferon. The intracellular level of Pr65 was not lowered in the interferon-treated cells, indicating that inhibition of viral protein synthesis was not responsible for inhibition of virus production. The present study suggests that interferon-mediated inhibition of retrovirus production, in general, is not a consequence of either a defective interaction between viral nucleoprotein cores and viral envelope proteins or a defect in the proteolytic processing of the gag polyprotein, since neither of these processes occurs during the morphogenesis of Gazdar particles and their production is nonetheless inhibited by interferon.

Enhancement of endogenous xenotropic murine retrovirus expression by tuftsin.

The exposure of a high-passage clone of Kirsten sarcoma virus transformed Balb/c (K-Balb) mouse cells to tuftsin (Thr-Lys-Pro-Arg) enhanced the expression of endogenous xenotropic retrovirus. The tetrapeptide increased the expression of virus that was infectious for rat, but not mouse, cells in a concentration-dependent fashion (0.001-1000 micrograms/ml). Increased virus expression could be achieved during short-term incubations (3-4 hr), with maximum enhancement occurring over longer time periods (16-18 hr). The enhancement of virus expression by tuftsin was proportional to the spontaneous release of virus. The infectivity of the enhanced virus was neutralized by goat anti-RLV gp 70 serum. Actinomycin D inhibited the induction of virus, suggesting that enhanced expression required de novo RNA synthesis. Tuftsin stimulated DNA, RNA, and protein synthesis in K-Balb cells during 16-hr incubations. Increased cellular proliferation was also seen at various time periods. The effects observed using K-Balb cells offer an opportunity to study the modulation of gene expression by tuftsin in a fibroblast culture system.

Effect of mouse interferon on retrovirus production by chronically infected rat cells.

Mouse interferon (IFN) inhibited retrovirus release by both mouse and rat cells with the same efficiency. However, the antiviral state developed more slowly in rat than in mouse cells, and after removal of IFN it also persisted for a longer time in rat than in mouse cells. Under conditions where IFN strongly inhibited virus production it had no effect on cell replication nor on cellular RNA or protein synthesis.

Murine xenotropic type C viruses. IV. Replication and pathogenesis of ducks.

The xenotropic (X-tropic) mouse type C virus (MuLV) and its pseudotype of murine sarcoma virus (MSV) were inoculated into several fertilized developing Pekin duck eggs. The development of the duck embryos was substantially reduced in those receiving the X-tropic viruses compared to eggs inoculated only with tissue culture medium. Infections virus was isolated from some of the adult animals; in others, evidence for integrated virus sequences in the tissues was noted. No specific pathology was found in the ducks that received X-trophic MuLV alone, but one duck developed multiple fibrosarcomas when inoculated at birth with the X-tropic virus pseudotype of MSV. Two ducks receiving X-tropic MuLv had signs of haematopoietic disorders. In addition, more virus-inoculated animals had evidence of hepatitis and encephalitis than control ducks. Antibody production to X-tropic MuLv was present in several ducks inoculated with virus either in embryo or at birth. Absence of antiviral antibodies was noted in those animals whose tissue contained replicating virus. These studies confirm the observations with X-tropic virus in tissue culture. They demonstrate in vivo that avian species are susceptible to infection by the mouse X-tropic virus and that their fibroblasts can be transformed by the X-tropic MuLV pseudotype of MSV.

Growth of murine sarcoma and murine xenotropic leukemia viruses in Japanese quail: induction of tumors and development of continuous tumor cell lines.

The BALB/c murine endogenous xenotropic leukemia virus pseudotype of m1 Moloney sarcoma virus [m1MSV(B-MuX)] was used to productively transform diploid Japanese quail embryo cells. The majority of newly hatched quail inoculated with m1MSV(B-MuX)-transformed quail embryo fibroblast cells rapidly developed tumors which were predominantly locally invasive fibrosarcomas, but metastases were observed in two birds. In two tumor-bearing quail, lymphosarcomas were observed in conjunction with fibrosarcomas. Quail inoculated with m1MSV(B-MuX) virus or quail embryo fibroblast cells infected with helper leukemia virus did not develop tumors. A cell culture derived from one quail tumor was shown to be oncogenic in newly hatched quail. Viruses produced by m1MSV(B-MuX)-infected quail cells or quail tumors had envelope properties of BALB-derived murine xenotropic leukemia virus as measured by host range, interference, and neutralization. Virus structural antigens, proteins with molecular weights of 30,000 and 70,000, were detected in tumors and tumor-derived cell lines by immunofluorescence and gel diffusion. Sera from tumored quail had high titers of type-specific BALB-derived murine xenotropic leukemia virus antibodies as determined by neutralization and immunoprecipitation. Antibodies to the putative m1 Moloney sarcoma virus mos gene product were not detected in sera from tumored or regressor quail.

Cell cycle-specific inhibition by retinoic acid of xenotropic murine retrovirus expression.

Several retinoids were examined for their capacity to block chemically induced expression of endogenous xenotropic retrovirus from Kirsten sarcoma virus-transformed BALB/c mouse cells. Retinoic acid (RA) was found to inhibit induction of virus by 5-iododeoxyuridine, cycloheximide, and histidinol; inhibition was concentration (10(-4) to 10(-6) M) and time dependent (1 to 7 hr) and not a consequence of cytotoxicity. Following a 6-hr treatment with 10(-4) M RA, [3H]thymidine and [3H]uridine incorporation into total cellular DNA and RNA was reduced 37 and 63%, respectively. Heteronuclear RNA synthesis was reduced 36 and 7% within 4 hr by 10(-4) and 10(-5) M RA, respectively, indicating that inhibition was not the result of a general transcriptional block. Using synchronized cells, it was found that 5 X 10(-5) M RA added in G1 phase and followed by cycloheximide or 5-iododeoxyuridine induction inhibited virus expression 60 and 84%, respectively. Little or no inhibition was observed when RA was added during S phase with the inducers or during G2 phase followed by inducers. Cells synchronized by mitotic arrest showed a RA-mediated restriction point in early-to-mid-G1 phase as indicated by a delay in the onset of DNA synthesis and an inhibition of virus induction during S phase. The results show the presence in Kirsten sarcoma virus-transformed BALB/c cells of a RA-sensitive G1 restriction point for cell progression and suggest that inhibition of retrovirus activation may be related to an extended G1 phase.

Viral susceptibility of skin fibroblasts from patients with Huntington disease.

Cultured skin fibroblasts from patients with Huntington disease (HD) and age-matched controls were tested for susceptibility to vesicular stomatitis virus (VSV) and transformation by Kirsten mouse sarcoma virus (KiMSV). The HD and control cells could not be distinguished on the basis of viral replication, plaque morphology, virus yield, or susceptibility to transformation by KiMSV. These findings suggest that the HD gene product, if expressed within peripheral tissue, does not selectively alter or interfere with viral replication.

Cellular proliferation in the spleen of DBA/2 mice infected with a myeloproliferative sarcoma virus(MPSV).

Cellular proliferation kinetics were investigated in the spleens of DBA/2 mice infected with a myeloproliferative sarcoma virus (MPSV) at three distinct phases of spleen growth: the rapid-growth phase, the slow-growth phase and the regression phase. Using injection of tritiated thymidine in vivo and autoradiographic techniques, we showed that most blast cells proliferate rapidly (cell cycle is equal to 10 h) even during the slow-growth phase. Very massive and rapid cell loss was found during the rapid-growth phase (cell loss factor phi = 80%). By comparing the decrease in specific splenic activity after in vivo injection of 125IUdR and 3HTdR, we were able to show a large reutilization of tritiated thymidine (R = 57%), visible less than 7 h after injection of isotopic DNA precursors. Thus, MPSV-infected spleen cells were shown, for the most part, to be short-lived cells: almost one-half mature into erythrocytes and the others rapidly die in situ.

Inheritance of susceptibility to the myeloproliferative sarcoma virus: effect of the Fv-2 locus and evidence for a myeloproliferative sarcoma virus resistance locus.

Myeloproliferative sarcoma virus (MPSV) causes a generalized stem cell leukemia with erythroid and myeloid hyperplasia in adult mice. MPSV also transforms fibroblasts. Mice congenic for the Fv-2 locus showed marked differences in susceptibility to MPSV according to the Fv-2 genotype. MPSV was injected into C57BL/6 Fvs and C57BL/6 Fv-2r mice congenic except for the Fv-2 locus. C57BL/6 mice with the Fvs genotype were much more susceptible to MPSV than were those with the Fvr genotype. Both DDD Fv-2r mice congenic with DDD Fv-2s mice except for the Fv-2 locus and DDD Fv-2s mice, however, were sensitive to spleen focus formation by MPSV. These data indicate that at least one additional resistance locus to MPSV is present in C57BL/6 mice but not in DDD mice. Both the Fv-2 locus and the putative MSPV resistance locus (loci) Mpsvr appear to be epistatic to either of the sensitivity loci. Fibroblast focus formation by MPSV was obtained well in C57BL/6 Fv-2r and C57BL/6 Fvs fibroblasts, indicating that the genes for MPSV resistance (Fv-2r and Mpsvr) were not operating in fibroblast cells. A model is proposed which may account for the differences in response of genetically different mice to MPSV and Friend spleen focus-forming virus.

Further studies on anti-tumor activity of adenovirus fibre protein on murine sarcoma tumours.

The present study demonstrates that subcutaneous injection of purified adenovirus 12 fibre protein (FP) to 4-week-old Balb/c mice, 4, 2 and 1 day(s) before intramuscular injection of murine sarcoma virus (MSV-M) or 2 and 1 day before and 1 day after MSV-M injection causes significant inhibition of tumour growth (P less than 0.001). The evidence suggests that treatment with FP leading to tumour inhibition is due to neither an interferon mechanism nor to inhibition of virus multiplication of FP in the reticuloendothelial system. It is postulated that immune functions, probably against MSV-induced cell antigens, might be the critical mechanisms underlying inhibition of tumour growth.

Host range of murine sarcoma virus, its host modification and phenotypic mixing with endogenous virus.

The M(MSV)AG-50 cells produce competent ecotropic sarcoma virus which is able to transform several rodent embryo cells and replicate in them. The expanded host range has shown that the virus acquired some host information which facilitate the transformation of heterologous cells. The transformed cells were XC positive and with transformed phenotype in vitro. The genome of mouse sarcoma virus from nonproducer rat liver cells could be rescued by xenotropic endogenous virus. The obtained virus has shown the augmented host range for various embryo cells and for some mammalian cell lines as well. The virus with xenotropic coat efficiently transforms the cells, yielding cells with transformed phenotype. The majority of this virus were phenotypically mixed virions, with minority of probably recombinant virus as suggested by XC test. The modification of the virus during the passage through heterologous cells is discussed.

Simian virus 40 and Moloney-murine sarcoma virus infection of bona fide mouse epithelium.

Moloney-murine sarcoma virus (Moloney-MSV) and simian virus 40 (SV40) were found to infect successfully pure cultures of epithelial cells established from mouse liver and mammary tissue. MSV infection resulted in transient morphological foci with persistent production of infectious virus. SV40 infection produced detectable levels of virus-specific T antigen in the cells but morphological transformants were not observed.

The isolation and characterization of a clonally related series of murine retrovirus-infected mouse cells.

Starting with cloned NIH 3T3 mouse cells we have isolated a series of related lines infected with the Kirsten murine sarcoma/leukaemia (MSV/MLV) virus complex. These lines exhibit all three possible infected cell phenotopes: (i) transformed MSV/MLV producers; (ii) non-transformed MLV producers; (iii) transformed non-producers. We have also selected non-transformed revertants from one of the non-producer clones. This series of lines allows the study of the expression of the virus genome against a constant background of cellular gene expression. We have further characterized the lines with regard to anchorage dependence of growth, tumorigenicity and the presence of a rescuable sarcoma genome. The non-producer clones are uniform in their transformed properties. The revertants contain rescuable sarcoma virus, biologically indistinguishable from the original transforming virus, implying that the reversion is due to a change in cellular rather than viurs genetic information.

Biological and molecular biological characterization of the virus progeny from transformed clones MuSV-124 and MuSV-349: evidence for MuLV-specific nucleotide sequences in the MoMuSV size class of RNA from MoMuSV-124.

The genetic information of MoMuSV-349 and MoMuSV-124, two clones of productively transformed TB cells, was distributed between two size classes of RNA (mol. wt. 2.9 x 10(6)) in the proportions of 5:1. Some preparations of MoMuSV-124 lacked the large RNA. The virions produced by both clones also contained all the nucleotide sequences of Moloney leukaemia virus and the ratio of MuSV:MuLV produced by the two clones differed markedly. The distribution of the sequences specific for Moloney murine leukaemia virus (MoMuLV) between the two size classes of RNA was studied using molecular hybridization to DNA probes complementary to and representative of: (i) the Moloney murine sarcoma virus (MoMuSV) RNA genome (mol. wt. 1.9 x 10(6)); (ii) those nucleotide sequences shared by MoMuSV and MoMuLV; (iii) nucleotide sequences specific for MoMuSV; (iv) nucleotide sequences specific for MoMuLV. The only detectable Moloney leukaemia virus-specific nucleotide sequences present in MoMuSV-124 virions were in the RNA of mol. wt. 1.9 x 10(6), whereas these sequences were detected in the RNA of mol. wt. 2.9 x 10(6) isolated from MoMuSV-349 virions. The biological properties of the replicating information in MoMuSV-124 suggest that, consistent with the small size of RNA, it is defective. whereas MoMuSV-349 produces virions containing an intact MoMuLV genome, competent for replication.

Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice.

Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transformation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.

Efficient production of mammalian RNA tumor viruses in serum-free culture medium allows rapid RNA subunit purification.

A wide variety of infected mammalian cell cultures were observed to produce high levels of RNA tumor virus particles in the absence of serum for at least 12 h. Virus production was measured by yields of 50-70 S virus RNAs isolated directly from serum-free culture media by chromatography on oligo(dT)-cellulose. Yields of RNAs from viruses produced in serum-free medium were comparable to yields obtained from purified viruses produced in serum-containing medium. Subunits of viral RNAs were thermally dissociated and separated by a new sedimentation system using sucrose gradients with resolving power (in the relevant size range) equivalent to that obtained with electrophoresis in polyacrylamide gels. RNA subunits isolated directly from serum-free medium appeared to be intact as judged by poly(A) content and resedimentation. The overall approach developed here represents dramatic savings in time and effort over previous ways of producing purified RNA subunits from tumor viruses.

Inhibition of murine sarcoma virus induced transformation in an adenovirus -- NRK system.

Cell transformation induced by murine sarcoma virus (MSV-M) is significantly inhibited (80--90%) in a clonal line of normal rat kidney (NRK) cells when they are infected with rat cell passaged adenovirus 12 (R-Ad12). No inhibition is seen when R-Ad12 is added simultaneously with, or 1 1/2 or 24 hr after, MSV-M infection, suggesting that inhibition occurs most probably intracellularly. There is also a direct correlation between the extent of focus formation and the concentration of R-Ad12 used. Concentrations of R-Ad12 used to inhibit cell transformation do not affect cell growth. Significant inhibition in foci (90--100%) is also evident in the first two subcultures of R-Ad12 infected NRK cells. The mechanism of the inhibition is not yet known.

Analysis of proteins of mouse sarcoma pseudotype viruses: type-specific radioimmunoassay for ecotropic virus p30's.

Murine sarcoma virus pseudotypes were prepared by infection of nonproducer cells (A1-2), which were transformed by the Gazdar strain of mouse sarcoma virus, with Gross (N-tropic), WN1802B (B-tropic), or Moloney (NB-tropic) viruses. The respective host range pseudotype sarcoma viruses were defined by the titration characteristics on cells with the appropriate Fv-1 genotype. Proteins from virus progeny were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bands present in both the 65,000- and the 10,000- to 20,000- molecular-weight regions of the gel distinguished the pseudotype viruses from their respective helpers. Furthermore, two protein bands were noted in the p30 region of murine sarcoma virus (Gross), one corresponding to Gross virus p30, and another of slightly slower mobility. However, since the mobility of the putative sarcoma p30 is nearly indentical to that of WN1802B, its presence could not be established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Type-specific radioimmunoassays for Gross virus p30 and for WN1802B p30 were applied for analysis of pseudotype preparations, and among several ecotropic viruses tested, only the homologous virus scored in the respective assay. By use of these assays, pseudotype viruses were found to contain only 8 to 48% helper-specific p30's; the remainder is presumably derived from the sarcoma virus.